Method and apparatus for separating biological materials

ABSTRACT

According to various embodiments, a system can be provided to separate undifferentiated cells and/or stromal cells from a whole tissue sample. The whole tissue sample can be any appropriate tissue sample obtained directly from a patient. The tissue sample can be obtained during a selected operating procedure for immediate or quick application or re-application to the patient. Accordingly, autologous cells can be obtained intraoperatively for application to a patient substantially soon after obtaining a whole tissue sample.

FIELD

The present disclosure is directed to a method and apparatus forseparating biological materials, and particularly to a system forseparating a selected fraction from a multiple-component biologicalmaterial.

BACKGROUND

This section provides background information that-is related to thepresent disclosure, but that is not necessarily prior art.

Various cellular (or biological) materials can be used to assist ahealing or recovery process in a human patient. Selected cell types,such as stromal cells, pluripotent or multipotent stem cells, or fullydifferentiated cells, can be applied therapeutically to the patient. Forexample, stem cells can be applied to an affected area of the patient,such as an area that may be damaged due to injury, chemotherapy, orradiation therapy, to assist in healing the area through differentiationof the stem cells and regeneration of the affected cells.

In performing a therapeutic procedure on a human patient usingundifferentiated cells, such as stem cells or stromal cells, theundifferentiated cells can be obtained from various sources, includingthe patient's own anatomy. Accordingly, certain autologous cells can beapplied to or injected into various portions of the anatomy of thepatient. Generally, a whole tissue or whole blood sample can be obtainedfrom the patient during a first procedure, selected cells can beseparated from the whole tissue or blood sample, and the selected,separated cells can be reapplied to or injected into the patient duringa subsequent procedure.

SUMMARY

This section provides a general summary of aspects of the invention, andis not a comprehensive description of its full scope or all of itsfeatures.

According to various embodiments, a system can be provided to separateundifferentiated cells, including stem cells and/or stromal cells, froma whole tissue or whole blood sample. The sample can be any appropriatetissue or blood sample obtained directly from a patient. The sample canbe obtained during a selected operating procedure with the object ofseparating undifferentiated cells from the sample for immediate or quickapplication to the patient. Accordingly, autologous undifferentiatedcells can be obtained intra-operatively for application to a patientrelatively soon after obtaining a whole tissue or whole blood sample.

Further areas of applicability will become apparent from the descriptionprovided herein. The description and specific examples are intended forpurposes of illustration only and are not intended to define the scopeof the claimed invention.

DRAWINGS

The drawings described herein are for illustrative purposes only ofselected embodiments and not all possible implementations, and are notintended to limit the scope of the present disclosure.

FIG. 1 is an elevation view of a separating system;

FIG. 2A is an exploded perspective view of a buoy system of theseparating system of FIG. 1;

FIG. 2B is a cross-section view of the buoy system of FIG. 1 in plane2B-2B;

FIG. 3 is an elevation view of a method of filling the separation systemof FIG. 1;

FIG. 4 is a perspective view of a centrifuge system;

FIG. 5A is an elevation view of an operation of the buoy system of theseparating system of FIG. 1 shown partially in section;

FIG. 5B is a detail view of an operation of the buoy system, shown insection, of the separating system of FIG. 1;

FIG. 6 is an elevation view of a fractionated whole tissue or wholeblood sample in the separation system of FIG. 1; and

FIG. 7 is an environmental view of an application of a selected fractionof the whole tissue or whole blood sample.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION

Example embodiments will now be described more fully with reference tothe accompanying drawings.

With reference to FIG. 1, a cell separating system 20 is illustrated.The cell separating system 20 can include a container 22 of anyappropriate configuration, such as a substantially cylindrical containerhaving a cylindrical outer wall 24 and closed by a substantiallycircular bottom wall 26 and a substantially circular upper wall orremovable cap 28. The container 22 extends along a longitudinal axis Aand the top wall 28 and the bottom wall 26 are positioned separate fromeach other along and substantially perpendicular to the longitudinalaxis A. It will be understood, however, that the container 22 can haveany appropriate cross-section, such as a polygonal cross-section, asquare cross-section, or any other appropriate shape.

The separating system 20 can include a buoy system 30 positionablewithin the container 22 and able to move generally axially along thelongitudinal axis A relative to the bottom wall 26 and the top wall 28.Buoy system 30 can be configured to contact, in a desired, selectedmanner, an inner wall surface 32 of the wall 24. For example, the buoysystem 30 can be configured to frictionally engage the inner wallsurface 32 while separating system 20 is in a substantially staticstate, and yet move axially relatively freely with respect to thecontainer 22 while separating system 20 is being centrifuged, asdescribed further below. According to various embodiments, the container22 can be formed of a material selected such that wall 24 can flexoutwardly and transversely to the longitudinal axis A, under a selectedforce. For example, as disclosed in U.S. Pat. No. 7,374,678, issued May20, 2008, incorporated herein by reference, the container 22 can bepositioned in a centrifuge and spun so that a large force, generallyseveral times that of gravity, is applied towards the bottom wall 26 ofthe container 22 so that the wall 24 flexes outwardly and container 22compresses. In the compressed state, the cylindrical outer wall 24 canexpand in diameter such that inner wall surface 32 can move radiallyaway from the buoy system 30. Consequent clearance between buoy system30 and inner wall surface 32 allows buoy system 30 to move axiallywithin the container 22 and relative to the inner wall surface 32.

The buoy system 30, as discussed in further detail herein, can includeor define a separation or collection volume 40 between an upper buoyportion or member 42 and a lower buoy portion or member 44. A withdrawalmember or tube 46 can interconnect a withdrawal port 48 with anextraction port 50 associated with the buoy system 30. The withdrawalport 48 can extend through the top wall 28 to an exterior of thecontainer and the extraction port 50 can communicate with and allowaccess to the collection volume 40 within the buoy 30. The withdrawalport 48 can include a connector, such as a luer connector 52, that canbe selectively capped or blocked with a cap or plug 54.

An introduction port 56 can also extend through the top wall or cap 28and communicate with an interior volume 57 of container 22. Introductionport 56 can also include a luer connector or an internal taper wall 58that can interconnect with an introduction syringe 60. A cap or plug 62can be selectively interconnected with the introduction port 56, after amaterial is introduced into the container 22, to cap or block luerconnector 58. Also defined through the top wall or cap 28 can be asecond fraction or plasma withdrawal port 64 that can include a luerlock or connector 65 and also be selectively capped or blocked with acap or plug 66. The second fraction or plasma withdrawal port 64 can beconnected with a port or bore defined through the cap 28 to withdraw orremove a material that is positioned within volume 57 between the buoysystem 30 and the cap 28 at any selected time, such as after aseparation step formed with the separation system 20.

With reference to FIGS. 2A and 2B, the buoy system 30 can include theextraction port 50, the upper buoy portion 42 and the lower buoy portion44. The upper buoy portion 42 can have legs or arms 70 extending axiallyfrom an annular ring or member 72 to contact an upper transverse surface74 of the lower buoy portion 44. The legs 70 can extend generallyparallel with the longitudinal axis A of the container 22. The legs 70can contact the upper surface 74 to help support the annular ring 72 ofthe upper buoy portion 42. The legs 70 can be fixed or connected to theupper surface 74 or only contact the upper surface 74 in a non-fixedmanner.

The legs 70 can be formed in any appropriate configuration or number,including two legs as illustrated in FIGS. 2A and 2B, four legs, anyappropriate number of legs, or any other appropriate structure, shape orconfiguration. For example, a colonnade can be formed near an outer edgeof the first buoy portion 42 and the second buoy portion 44 to assist inholding the two portions 42, 44 apart. Regardless, the legs 70 cansupport the first buoy portion 42 at a selected distance from the secondbuoy portion 44. In yet another configuration, legs 70 can be omitted.

The annular member 72 can connect to spokes or extension members 76extending radially from a central post or tube 78. The spokes 76 extendgenerally perpendicular to the axis A. The spokes, however, can extendat an angle relative to the central axis A for reduced drag, etc. Thetube 78 defines an internal bore 80 that can communicate with a passageor internal bore 82 of the withdrawal port 50 to permit removal ofmaterial from the collection volume 40 of the buoy system 30. Thecentral tube 78 includes an external wall 84 that extends axially fromradial spokes 76 at a center or middle of the annular ring 72 to contactthe lower buoy portion 44.

A connecting or fusion portion 86 of the central post or tube 78 canextend into and be fixed to the lower buoy portion 44 in an internalwell or bore 90. The connecting portion 86 of the post 78 can beadhered, molded, fused, snap fitted, press fitted or interference fittedwith the second buoy portion 44. The connection portion 86 can hold thefirst buoy portion 42 to the second buoy portion 44. Accordingly, thecentral post 78 can also assist legs 70 in holding the annular ring 72 adistance from the lower buoy portion 44 to define the collection volume40. Alternatively, central post 78 can hold the annular ring 72 spacedfrom lower buoy portion 44 in the absence of legs 70.

Fins or raised portions 92 can extend upwardly from the surface 74 ofthe lower buoy portion 44. The fins 92 can include a ridge or notchedportion 94. The central post 78 also can be fixed to the fins 92 toassist in fixing or holding the first buoy portion 42 a selecteddistance from the second buoy portion 44.

Lower buoy portion 44 can include upper surface 74, an annular orexterior wall 102 and a lower surface 45. The upper surface 74 of thelower buoy portion 44 can be conical and angled or inclined downwardlyand inwardly from an outer circumferential edge 100 defined by theintersection or connection of the annular or exterior wall 102 and theupper surface 74. In an imaginary plane that includes longitudinal axisB of the central post 78 and that intersects the upper surface 74, axisB and the line of intersection of upper surface 74 subtend an angle a.The angle a can be any appropriate angle such as about 45° to about 90°,preferably about 75°. Alternatively, upper surface 74 can be concavelyspherical rather than conical.

The central portion of the lower buoy portion 44 can define a sump orlow portion 103 where a volume of material can be collected, asdiscussed further herein. The sump 103 can assist in forming a pellet ofa selected portion of material, such as a cell fraction. The centralpost 78, which defines an axial bore 80, can also include a throughpassage that connects bore 80 in communication with an externalreceiving port 104 defined by outer wall 84 of post 78. The port 104 canbe positioned in or at the bottom of the sump 103 to allow withdrawal ofthe collected material through the tube 78 and, in turn, throughextraction port 50, withdrawal tube 46, and the withdraw port 48.

Positioned around the central post or tube 78 can be a valve system 110.The valve system 110 can include a sealing member 112 made of anappropriate material, such as a silicone rubber material. The sealingmember 112 can be configured as a disc or washer including a centralaperture, such as a round hole 113, to receive and fit around thecentral tube 78. A holding or valve actuation member 116 can include anupper apertured disc or washer 117 that is similar in surface area tothe sealing member 112 and a mounting or holding cylinder or tube 118that extends downwardly substantially perpendicularly from disc orwasher 117. Holding cylinder 118 and washer 117 receive and fit aroundcentral tube 78 below sealing member 112. Holding cylinder 118 can alsocontact and be supported by the ledges 94 of fins 92 above surface 74,thereby leaving a circumferential clearance around tube 78 relative tofins 92 and holding cylinder 118 to provide communication among thewedge shaped spaces between fins 92. Briefly, the sealing member 112 andthe disc 117 can form a flapper valve against the first buoy portion 42.The upper disc 117 of holding member 116 can bend to allow the sealingmember 112 to move away from the first buoy portion 42, as discussedfurther herein.

The height of the holding cylinder 118 can allow it to contact the ledge94 and support the disc 117 at an appropriate elevation to hold thesealing member 112 against a bottom surface 120 of the annular member 72when no opening force is acting on the valve portion 110. The spokes 76can interconnect the annular member 72 with the central tube or post 78but also form passages 122 (i.e. openings) between the spoke member 76.The passages 122, when opened by the valve system 110, allow material tomove from an area above or exterior to the separation buoy system 30into the collection volume 40.

The valve system 110 can be formed as one piece that includes both thesealing member 112 and the valve actuation member 116. For example, thesealing member 112 can be a flexible material, such as a siliconerubber, spread or applied to the disc 117 of the actuation portion 116.Alternatively, the sealing member 112 can be a separate piece that sfixed to the valve disc 117 with an adhesive or other connectingmechanism. Further, alternatively, the sealing member 112 can beseparate from the valve actuation member 116 and only held in place withthe biasing force of the valve actuation member 116. In other words, thesealing member 112 can be manufactured separate from the actuationmember 116 or assembled together as the valve system 110.

The valve actuation member 116 can be formed of a material that can flexwhen a force is applied to it, such as a centrifugal force or pressuredifferential force, as discussed in detail herein. The material selectedfor the valve actuation member 116 can include acrylic, polycarbonate,and any other appropriate resilient and substantially inert material Thevalve actuation member 116 can be formed of a resilient, yet flexiblematerial. Thus, the actuation member 116 can provide a biasing orclosing force on the sealing member 112 against the first buoy portion42. The valve actuation member 116, therefore, can rebound from an openposition to bias the valve 110 in the closed position.

The buoy system 30 can be made of any appropriate material and can bemade of a material selected according to the biological material to beplaced and separated in the separation system 20. The buoy system 30,however, can be made of materials having a mean density in the range ofabout 1.03 g/ml to about 1.10 g/ml, preferably in the range of about1.045 g/ml to about 1.07 g/ml. The density or specific gravity of thebuoy separation system 30 can be selected to position the collectionvolume 40 at an area or position within the separation container 20relative to the material positioned in the separation system 20. Thespecific gravity of the buoy system 40 can be selected to move to aselected interface of two or more fractions of a separated multiplecomponent material during separation.

For example, the second buoy portion 44 can be made of a single materialor multiple materials and the upper buoy portion 42 can also be made ofa single material or multiple materials. The density can be selected byselecting appropriate portions of the materials from which each of themembers is made. Selecting the density of the material from which eachof the elements of buoy system 30 is made, together with selecting therelative volumes, locations and dimensions of the elements, can assistin achieving a selected placement of the buoy system 30 within thematerial. The materials can also be selected to be substantiallynon-reactive to the material being separated in the system 20.

With reference to FIGS. 3-7 a method of using the separation system 20is illustrated. Initially, a whole tissue sample 140 (e.g. bone marrowaspirate, whole blood, a combination of blood and bone marrow) ispositioned within the separation system 20 via introduction from thefilling syringe 60. The buoy system 30 can be positioned near the bottomwall 26 of the separation container 22 during filling. The buoy system30 can move during a centrifugation stage, as discussed further herein.

The whole tissue sample 140 positioned in the separation system 20 canbe any appropriate whole tissue sample. For example, the whole tissuesample can include whole blood, bone marrow aspirate or a mixture ofwhole blood and bone marrow aspirate. The whole tissue sample caninclude that described in U.S. Pat. No. 7,374,678, incorporated hereinby reference above. The whole tissue same 140 positioned within theseparation system 20, however, can be separated into selected portionsor fractions and selected fractions can be withdrawn from the separationsystem 20.

The separation system 20 can then be positioned in a centrifuge device150, as illustrated in FIG. 4. The centrifuge device 150 can include acentrifuge member or rotor 152 with separation wells that can hold theseparation system 20 and appropriate blanks or other separation systems.The separation centrifuge 150 can include a centrifuge similar to thecentrifuge sold by Biomet, Inc. with the GPS® Platelet SeparationSystem. The separation system 20 can be spun in the centrifuge to directa centrifugal force along the longitudinal axis A of the separationsystem 20 towards the bottom wall 26. During centrifugation, thematerials of a multi-component material can separate based on specificgravity and density of the components. As understood by one skilled inthe art, denser materials will move towards the bottom wall of theseparation system 20 and lighter materials move towards the top wall 28.

As illustrated in FIGS. 5A and 5B, during centrifugation, as densematerials move towards the bottom wall 26, the buoy system 30 can movetowards the top wall 28 of the separation system 20. As the buoy system30 moves towards the top of the separation container 28, generally inthe direction of arrow X, the centrifugation force and the force of amaterial pressing on the valve system 110 can bend edges, such as outeror peripheral edges 112 a and 117 a of the valve system 110. When theedges or outer portions 112 a, 117 a bend or move away from the undersurface 120, the valve is opened or unsealed. The sealing member 112unseals from or moves to the open position relative to the annularmember 72, such as the bottom portion 120 of the annular member 72. Asillustrated in FIG. 5A and in detail in 5B, when the edges 112 a, 117 aof the valve system 110 are bent, material can be move in the directionof arrow Y through the voids or passages 122 between the spokes 76 andinto the collection volume 40.

Initially, the valve system 110 can be bent by the centrifugal forcesand movement of the whole tissue sample 140 towards the collectionvolume 40. Then the valve 110 can remain in the open position, bent,during centrifugation due to the density difference between the denserflexible material of discs 112 and 117 and the less dense biologicalmaterial, such as plasma.

Also, as discussed above, the separation container 22 can flexoutwardly, allowing the buoy system 30 to move within the container 22during centrifugation. The buoy system 30, having the selected densityor specific gravity, can move within the separation container 22 to aselected interface of appropriate materials or fractions of the wholesample 140 that is being separated by the centrifugal force. Due to theflex of the container 22 a space can form between the buoy system 30 andthe interior wall 32. Thus, material can also move in the direction ofY′ around the buoys system 30. The lower buoy portion 44 can include aconical lower surface 45 to assist in movement of the buoy system 30away from the lower wall 26.

As illustrated in FIG. 6, once centrifugation has slowed sufficiently,or stopped, the valve system 110 can close with the biasing force of theactuation member 116, in the absence of the opening or external force(e.g. the centrifugal force). The buoy system 30, due to its selecteddensity, can allow the collection volume 40 to be positioned at aselected interface within the separated sample 140. The separated wholetissue sample 140 can include at least a first fraction 140 a that canbe closest to the top wall 28 of the separation container 22 and asecond fraction 140 b that can be within the collection volume 40 of thebuoy separation system 30. The second fraction 140 b can flow around thebuoy system 30 when the container 22 flexes. The whole sample 140 canalso be separated to include a third fraction 140 c that can be closestto the bottom wall 26 of a separation container 22.

After the centrifugation has stopped, the valve system 110 can close orseal the collection volume 40 from the first fraction 140 a.Accordingly, during extraction or removal of the second fraction 140 bwithin the separation or collection volume 40, the first fraction 140 awill not interfere or mix with the second fraction 140 b. Because thevalve system 110 can assist in maintaining a physical separation of theselected fraction or second fraction 140 b from the other fractions, theseparated material can be maintained substantially pure and have a highyield.

Once the separation has concluded, an extraction withdraw syringe orsystem 160 can be used to withdraw the material or selected fraction 140b from the separation volume 40 via the interconnection of thewithdrawal port 48 via the tube 46, the extraction port 50, the centralpost or tube 78, and the withdrawal hole 104. Once the material iswithdrawn from the separation system 20, a user 170, such as a surgeon,can apply the material to a patient 180 in any appropriate manner. Forexample, the extraction withdrawal syringe 160 can be fitted orinterconnected with a needle 182 to allow injection of the selectedfraction into the patient 180. The selected fraction 140 b, as discussedabove, can include undifferentiated and/or stromal cells that can beapplied to the patient 180 for selected purposes, such as tissuere-growth, healing, or other appropriate purposes.

Thus, the separation system 20 can allow for an introduction ofautologous cells to the patient 180. The whole tissue sample 140 can bewithdrawn from the patient 180 with the delivery syringe 60 during asingle operative procedure. The centrifuge system 150 can be positionedin an operating room or near an operating room for appropriately timedseparation of the whole tissue sample to allow for extraction withdrawalof the selected fraction 140 b.

The foregoing description of the embodiments has been provided forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention. Individual elements or feature ofa particular embodiment are generally not limited to that particularembodiment, but, where applicable, are interchangeable and can be usedin a selected embodiment, even if not specifically shown or described.The same may also be varied in many ways. Such variations are not to beregarded as a departure from the invention, and all such modificationsare intended to be included within the scope of the invention.

1. A separation system to separate a multiple component material into atleast two fractions, comprising: a separation container defining avolume within an interior wall, a first end wall, and a second end wall;a buoy separation system in the volume operable to move to a selectedinterface of the multiple component material under the influence of aforce, the buoy separation system including; a first buoy portion havinga passage between a first side and a second side of the first buoyportion; a second buoy portion at a selected distance from the secondside of the first buoy portion, wherein the selected distance defines acollection volume between the second side of the first buoy portion andthe second buoy portion; a valve assembly having a sealing portion and aflexible portion, wherein the flexible portion holds the sealing portionagainst the first buoy portion to seal the passage in the first buoyportion to separate the collection volume from a volume exterior to thecollection volume on the first side of the first buoy portion; whereinthe valve assembly is operable to be unsealed from the first buoyportion upon the application of a force to allow a selected fraction tomove into the collection volume.
 2. A separation system of claim 1,further comprising: a centrifuge system having a centrifuge bucketoperable to hold the separation container and spin the separationcontainer around a central axis of the centrifuge bucket and apply acentrifugal force along a longitudinal axis of the separation container.3. The separation system of claim 2, further comprising: a withdrawalport extending through the first end wall of the separation container;and a connection tube interconnecting the withdrawal port and thecollection volume between the first buoy portion and the second buoyportion to allow withdrawal of the stromal cells when the valve assemblyis sealed; wherein the buoy separation system is operable to moverelative to the separation container based upon a force imparted on thebuoy separation system by the multiple component material when theseparation container is centrifuged in the centrifuge system.
 4. Theseparation system of claim 1, wherein the sealing portion and theflexible portion are a separate and distinct sealing member and flexiblemember moveable relative to one another; wherein each of the sealingmember and the flexible member have an inner wall defining a passage tofit the valve assembly around a central tube extending from the firstbuoy portion into the collection volume; wherein at least a portion ofthe flexible member is fixed relative to the first buoy portion.
 5. Theseparation system of claim 4, an extraction port formed through an outerwall of the central tube to allow withdrawal access to the collectionvolume.
 6. The separation system of claim 5, wherein the second buoyportion has an upper wall that defines a central sump to collect apellet of stromal cells.
 7. The separation system of claim 4, wherein aperipheral edge of the flexible member moves to allow the sealing memberto unseal from the first buoy portion.
 8. The separation system of claim7, wherein the flexible member includes a first portion that iscantilevered and flexes relative to a second portion of the flexiblemember.
 9. The separation system of claim 1, wherein the interior wallof the separation container substantially defines a cylindrical volume;wherein the first buoy portion includes an annular member extendingaround a central post and a spoke member extending from the central postto the annular member; wherein the spoke and the annular member definethe passage; wherein the valve assembly contacts the first side of thefirst buoy portion substantially adjacent the collection volume to sealthe passage through the first buoy portion.
 10. A separation systemincluding a buoy separation system to separate a multiple componentmaterial into at least a first fraction, the buoy separation systemcomprising: a first buoy portion having a passage between a first sideand a second side of the first buoy portion; a second buoy portion at aselected distance from the second side of the first buoy portion,wherein the selected distance defines a collection volume between thesecond side of the first buoy portion and the second buoy portion; avalve assembly having a sealing member and a flexible member, theflexible member having a dimension great enough to extend substantiallythe entire selected distance when contacting the second buoy portionwherein the flexible member holds the sealing member against the firstbuoy portion to seal the passage in the first buoy portion thusmaintaining a separation of the collection volume from a volume exterioron the first side of the first buoy portion; wherein the valve assemblyis operable to be unsealed from the first buoy portion upon theapplication of a force to allow the first fraction to move into thecollection volume.
 11. The separation system of claim 10, furthercomprising: a separation container having a volume defined by aninterior wall, a first end wall, and a second end wall.
 12. Theseparation system of claim 10, wherein the sealing member is fixed tothe flexible member.
 13. The separation system of claim 11, furthercomprising: a withdraw port extending through the first end wall; anextraction port extending from the first buoy portion; a tubeinterconnecting the withdraw portion and the extraction portion; and atube extending from the extraction portion into the collection volume.14. The separation system of claim 13, wherein the tube has an exteriorwall that, at least in part, fixes the first buoy portion relative tothe second buoy portion the selected distance to define the collectionvolume between the first buoy portion and the second buoy portion;wherein a first end of the tube extends to the first buoy portion and asecond end of the tube extends to a sump defined by an upper wall of thesecond buoy portion.
 15. The separation system of claim 13, wherein theupper wall of the second buoy portion is formed at an angle relative toan axis of the tube; wherein the angle is about 45 degrees to about 90degrees.
 16. The separation system of claim 10, wherein the flexiblemember includes a first disc portion extending radially from a centralhub portion, wherein the central hub portion extends substantially theentire selected distance; wherein the sealing member is an annularmember operable to be pressed against the first buoy portion by theflexible member.
 17. The separation system of claim 16, wherein theflexible member is operable to apply a force between the second buoyportion and the sealing member to seal the passage of the first buoyportion.
 18. The separation system of claim 17, wherein the first buoyportion includes a spoke extending from a tube to an inner wall of anannular portion; wherein the passage is defined at least in part by anouter wall of the spoke and the inner wall of the annular portion.
 19. Amethod of separating cells from a whole tissue sample, comprising:positioning a collected whole tissue sample in a separation containersystem; applying a centrifugal force to the collected whole tissuesample positioned in the separation container; moving a buoy separationsystem during the application of the centrifugal force and with amaterial force of the collected whole tissue sample; opening a valvethrough a first buoy portion of the buoy separation system to allow acell fraction to enter a collection volume between the first buoyportion and a second buoy portion; and closing the valve at thecessation of the application of the centrifugal force to form a physicalbarrier between the collection volume and a volume exterior to the firstbuoy portion; wherein opening the valve includes applying an externalforce and bending a flexible portion of the valve to move a sealingportion of the valve out of contact with the first buoy portion; whereinclosing the valve includes removing the external force from the flexibleportion and the flexible portion rebounding to move the sealing portioninto contact with the first buoy portion.
 20. The method of claim 19,wherein the external force is, at least in part, the centrifugal force;wherein opening the valve occurs due to a pressure differential betweenthe volume exterior to the collection volume and the pressure within thecollection volume; and maintaining the valve in an open position duringthe application of the centrifugal force due to a density of the valvebeing higher in the collection volume than the density of the wholetissue sample.
 21. The method of claim 19, further comprising:collecting bone marrow as a whole tissue sample.
 22. The method of claim19, further comprising: collecting whole blood as the whole tissuesample.
 23. The method of claim 19, wherein opening the valve includes,applying the external force to a flexible portion fixed relative to atleast a portion of the buoy separation system; wherein applying theexternal force to the flexible portion bends the flexible portion tomove the flexible portion away from a passage defined through at least aportion of the buoy separation system; wherein bending the flexibleportion further removes a force applied against the sealing portion thatseals a passage through the first buoy portion.
 24. The method of claim23, wherein applying the external force to the flexible portion,includes applying the external force to a flexible member to bend afirst portion of the flexible member relative to a second portion of theflexible member; wherein when the first portion is in the bentconfiguration the sealing portion moves to an open position.
 25. Themethod of claim 19, wherein positioning the collected whole tissuesample in a separation container includes positioning the collectedwhole tissue sample in a container containing the buoy separation systemwith the valve biased in a closed position, wherein opening the valveincludes applying a force to overcome the bias force of the valve in theclosed position.
 26. The method of claim 25, wherein closing the valveincludes removing the external force to allow the biasing force to bethe main force to close the valve.